Project Summary/Abstract Enzymes are well known for catalyzing chemical transformations with exquisite specificity and selectivity under environmentally benign conditions. Thus, there is a continuing need for the development of new enzymes that can effect critical synthetic transformations. One important type of reaction that is absent from the current catalytic repertoire of biology is the formation of carbon-boron bonds. Organoboron molecules have wide applications in organic synthesis, cancer therapy, and medicine. This prevalence of organoborons necessitates the development of new methods for introducing boron into molecules with high efficiency and selectivity. The focus of this proposal is to generate the first borylating enzymes and use them to expand the chemical space of biocatalysis to include carbon-boron bonds. The specific aims are: (1) 1: Develop the first enzymatic borylation via heme-protein-catalyzed iron-carbenoid insertion into B-H bonds. (2) Expand the scope of diazo compounds for the biocatalytic B-H insertion reaction. (3) Mechanistic and Structural Studies of the Optimized Borylation Enzymes. These aims will be initiated using the large number of existing mutants of various heme proteins in the Arnold Lab followed by directed evolution. Initial hits have been identified which provide a good starting point for directed evolution. We will perform detailed mechanistic and structural studies of the enzymes developed in this project, which will reveal the molecular basis of the improved activity and selectivity brought by the beneficial mutations. This knowledge is important for the further development of enzymatic borylation reactions, and can also be extended to other carbene-transfer reactions. The completion of these aims will open the door for new, highly selective, and sustainable strategies for synthesis of important, hard-to-make boron-containing molecules of biological and biomedical relevance.